β-Endorphin (an endogenous opioid) inhibits inflammation, oxidative stress and apoptosis via Nrf-2 in asthmatic murine model

Asthma, a chronic respiratory disease is characterized by airway inflammation, remodelling, airflow limitation and hyperresponsiveness. At present, it is considered as an umbrella diagnosis consisting several variable clinical presentations (phenotypes) and distinct pathophysiological mechanisms (endotypes). Recent evidence suggests that oxidative stress participates in airway inflammation and remodelling in chronic asthma. Opioids resembled by group of regulatory peptides have proven to act as an immunomodulator. β-Endorphin a natural and potent endogenous morphine produced in the anterior pituitary gland play role in pain modulation. Therapeutic strategy of many opioids including β-Endorphin as an anti‑inflammatory and antioxidative agent has not been yet explored despite its promising analgesic effects. This is the first study to reveal the role of β-Endorphin in regulating airway inflammation, cellular apoptosis, and oxidative stress via Nrf-2 in an experimental asthmatic model. Asthma was generated in balb/c mice by sensitizing with 1% Toulene Diisocyanate on day 0, 7, 14 and 21 and challenging with 2.5% Toulene Diisocyanate from day 22 to 51 (on every alternate day) through intranasal route. β-Endorphin (5 µg/kg) was administered through the nasal route 1 h prior to sensitization and challenge. The effect of β-Endorphin on pulmonary inflammation and redox status along with parameters of oxidative stress were evaluated. We found that pre-treatment of β-Endorphin significantly reduced inflammatory infiltration in lung tissue and cell counts in bronchoalveolar lavage fluid. Also, pre-treatment of β-Endorphin reduced reactive oxygen species, Myeloperoxidase, Nitric Oxide, Protein and protein carbonylation, Glutathione Reductase, Malondialdehyde, IFN-γ, and TNF-α. Reversely, β-Endorphin significantly increased Superoxide dismutase, Catalase, glutathione, Glutathione-S-Transferase, and activation of NF-E2-related factor 2 (Nrf-2) via Kelch-like ECH-associated protein 1 (Keap1), independent pathway in the lung restoring architectural alveolar and bronchial changes. The present findings reveal the therapeutic potency of β-END in regulating asthma by Keap-1 independent regulation of Nrf-2 activity. The present findings reveal the therapeutic potency of β-Endorphin in regulating asthma.

anterior pituitary gland from their precursor protein, proopiomelanocortin (POMC) which is further posttranslationally processed by a series of enzymatic activity giving rise to number of other peptides as ACTH, β-LPH, α-MSH 22,23 . It plays an important role in development of hypothalamus concerned non-sympathic and paracrine communication of brain message, central and peripheral analgesia and development of hyperalgesia 24 . Till several years, β-END was considered as a neuropeptide due to its multiple activities as neurotransmitter and neuromodulators but recent studies suggests that upon appropriate stimulation its synthesis and release also occurs in immune cells 25,26 . Immune cells are reported to possess mRNA transcript for POMC gene and hence are capable of forming different POMC derived peptides 26 . Strong evidence illustrates the key role of µ-opioid Receptor (MOR), δ-opioid Receptor (DOR) and κ-opioid Receptor (KOR) located on nervous system and immune cells and its increased expression in analgesia and stressful conditions. Studies reveal the tendency of β-END produced by immune cells to bind with strong affinity to MOR apart from DOR and KOR thereby producing anti-inflammatory cytokines such as IL-18, IL-10, IFN-γ and further impose analgesic effect 27 . Consequently, β-END synthesis from both CNS and immune system (cells) might be subjected to many regulatory inputs in several pathophysiologies. A recent study elucidates the role of β-END inhibiting the inflammatory response of bovine endometrial epithelial and stromal cells through DOR in vitro 28 . Investigation supporting the role of β-END as anti-inflammatory and anti-oxidative in airway disease is still lacking. Hence, the present study aims to evaluate the effect of β-END on the oxidative markers, cellular apoptosis by targeting Nrf-2 gene and its role in regulating inflammation in TDI induced asthma.
Result β-END attenuated airway inflammationin BALF. Inflammation in the airways and lungs parenchyma plays a central role in asthma. Inflammation was observed in the BALF as a mean of leukocyte recruitment to the lungs. Total cells were counted in BALF to evaluate cellular infiltration into lungs. Remarkable increase in the number of total leukocyte recruitments were observed in asthmatic experimental mice as compared to the control mice while administration of β-END inhibited the inflammatory cell accumulation to the lungs thereby reducing the inflammation (Fig. 1A). Among the four different categories of leukocytes the cellular profile of the recruited cells mainly included the macrophages and neutrophils as observed in the cytospin slides (Fig. 1D,E). TDI-induced mice showed an increased number and percentage of both neutrophils and macrophages in asthmatic mice TDI-OA as compared with normal mice while β-END administration significantly decreased both macrophages and neutrophils coun substizing the anti-inflammatory activity (Fig. 1B,C).

β-END regulates the TAS and TOS.
In the present experiment, a profound oxidant/antioxidant imbalance was observed in TDI induced asthmatic mice. The result showed an increase in TOS and continuous decrease in TAS when compared to the control mice. Administration of β-END downregulated the oxidant production ( Fig. 2A) and upregulated the antioxidant status (Fig. 2B) thereby recovering the balance of oxidant and antioxidant. No effect of olive oil and acetone (TDI solvent) was observed in the vehicle group when compared to normal. β-END inhibited total ROS, MPO activity, and NO level. The level of specific markers of oxidant stress such as intracellular ROS and MPO activity was significantly increased in TDI-treated mice compared with the normal mice. Conversely, a significant decrease in the ROS levels ( Fig. 3A) and MPO activity (Fig. 3B) was observed with the administration of β-END (5 µg/kg) when compared with TDI inhaled group. Nitrite level was measured as a marker of NO production. TDI-exposed group showed a significant increase in nitrite level as compared to the normal group whereas β-END significantly inhibited the nitrite level marking the decrease in NO production as compared with the TDI group (Fig. 3C). However no significant effect of vehicle was observed in ROS, MPO and NO level. β-END attenuated the protein content and its carbonylation. PC formation is an indicator of oxidative damage to protein. Significant increase in the concentration of total protein and PC was observed in the BALF as well as lung homogenate of TDI induced mice as compared to the control group while β-END (5 µg/kg) treatment significantly attenuated the total protein (Fig. 4A) and PC content (Fig. 4B) in BALF as well as in lung. No significant effects of acetone and olive oil (vehicle) were observed in the vehicle group. β-END regulated SOD, Catalase, GPx, GR, GSH and GST activity. As depicted in Fig. 5A and B, TDI induction significantly reduced the activities of SOD and Catalase, the main antioxidant enzymes as compared with those in control group. However, administration of β-END through nasal route restored the activity of SOD and Catalase to normal. Pre-treatment of β-END 1 h before TDI exposure also substantially restored the activity of GPx, GR, GSH and GST (in Fig. 5C-F) which were markedly modulated by TDI exposure. MDA level was regulated by β-END pre-treatment. MDA was measured in lung homogenate of different group as a biomarker of lipid peroxidation. It is assumed that increase ROS level leads to increase MDA level, an important biomarker released from the oxidation and decomposition of polyunsaturated fatty acids. After the last challenge, MDA level was significantly upregulated in the TDI group compared to the control mice (Fig. 6). β-END treatment by intranasal route significantly reduced the level of MDA compared with TDItreated mice (p < 0.05).  : TDI inhalation leads to the increase in recruitment of inflammatory cells when compared with the normal group as observed by total cell count whereas β-END significantly inhibited the recruitment of inflammatory cells The recruited cells in TDI group mainly include the increased number and percent of macrophages and neutrophils as enumerated in Differential cell count, Cellular Percentage, cytospun slides which were significantly inhibited by β-END. The red arrow represents neutrophils and the yellow arrow shows macrophages. Results are represented as means ± SEM (*p < 0.05 Control vs. TDI group and #p < 0.05 TDI vs. β-END group). www.nature.com/scientificreports/ Regulation of cytokines by β-END in TDI-exposed mice. As presented in Fig. 7A and B, a significant increase in TNF-α and IFN-γ was observed by TDI induction as compared with the normal group whereas β-END significantly (p < 0.05) downregulated TNF-α and IFN-γ as compared to TDI group. However, no effect of the vehicle was observed in both the cytokine.   Table 1. Each subpopulation was expressed as a percentage of the total population of granulocytes. Early and late apoptotic cells were significantly increased in asthmatic mice ( Fig. 8A and B) whereas administration of β-END significantly decreased the percentage of both early and late apoptotic cells thereby, maintaining the live cells. No significant changes were observed in necrotic cells in either of the groups.

β-END inhibits infiltration of cells and lung injury.
H&E staining was performed to further evaluate the morphometric pathological changes in the lung tissue in each group in terms of bronchoconstriction, inflammation, lung injury and emphysema. Control group showed normal lung architecture marked by no constriction of bronchioles and inflammation in airways and pulmonary blood vessels (Fig. 9). Also, there was no alveolar destruction representing no emphysema. The TDI-induced group showed bronchoconstriction ( Fig. 9) and increased inflammatory cell infiltration in alveolar areas (black arrow), peribronchiolar area (red arrow) and pulmonary blood vessels (blue arrow), alveolar destruction (airspaces enlarged) representing lung injury. β-END intranasal administration restored the normal architecture of the lungs where reduced bronchi-constriction, reduced inflammations (peribronchiolar, alveolar and pulmonary blood vessel) were observed (Fig. 9). β-END also improved the alveolar destruction caused by TDI induction (Fig. 9). Vehicle represented the normal architecture and was comparable to the control.
β-END regulated the expression of cytosolic and nuclear Nrf-2 and keap-1. Western blotting was used to examine Cytosolic and nuclear Nrf-2 and keap-1 expression in lung homogenates. In TDI-induced groups, a significant increase in cytosolic Nrf-2 and decrease in nuclear Nrf-2 was observed as compare to the control which was restored in β-END pre-treated groups ( Fig. 10A and B; supplementary file). Further, Keap-1 expression was found to be similar in TDI and β-END group representing no role of Keap-1 in Nrf-2 activation. Vehicle group showed similar results which was comparable to control.

Discussion
β-END, an endogenous opioid peptide, and morphine is a well-known analgesic which modulates pain. Till date, no study supports the role of β-END on airway inflammation regulating oxidative stress. Therefore, the present study has been proposed to investigate the therapeutic role of β-END on inflammation, oxidative and antioxidative mediators preventing lung injury via Nrf-2 expression in TDI provoked asthmatic model. Chronic exposure to TDI has been associated with the onset of asthmatic symptoms including airway inflammation, free radical generation, and release of chemical mediators as TNF-α and IFN-γ 29,30 . Although, airway inflammation is considered as the major hallmark of asthma including TDI-OA; oxidative stress has also been established to play an important role in its pathogenesis. Inflammation in asthma is characterized by inflammatory cell infiltration and their activation in the lung tissue, where macrophages and neutrophils are the most prominent effectors cells 31 . The present study was also consistent with previous studies where a significant increase in the recruitment of immune cells (including macrophages and neutrophils) was observed in TDIinduced asthmatic mice leading to inflammation 32 . Further, our finding reveals the protective effect of β-END on the recruitment of immune cells inhibiting the recruitment of macrophages and neutrophils.
The inflammatory mechanism is thought to introduce high ROS levels in the lung initiating oxidative stress which signifies the state of imbalance between the generation of oxidants (free radicals, ions, and reactive metabolites collectively called as ROS) and the availability of endogenous antioxidants defense system to eliminate ROS 33,34 . ROS act as a double edge sword in regulating redox status in immune cells 35 . Previous studies have reported the role of ROS resulting oxidative stress play a pivotal role in apoptosis of resident cells 36 . Also, several www.nature.com/scientificreports/ studies have evident the pivotal role of macrophages and neutrophils in the pathogenesis of asthma thereby generating ROS and RNS in the lungs inflicting macromolecules as protein denaturation, lipid peroxidation and DNA damage 37 . In the present study, increased ROS production represents the generation of free oxidants leading to apoptosis (early and late) of BALF cells which was substantially reduced by intranasal administration of β-END. The result suggests that recruited inflammatory cells (macrophages and neutrophils) may be the major contributor for ROS production whereas administration of β-END suppressing the recruitment of macrophages and neutrophils effectively declined the ROS content. Peroxidases as EPO and MPO released from activated cells are another major source for ROS during inflammation which catalyzes the generation of hypohalous acids responsible for deleterious effect on lung tissue amplifying inflammatory response 38 . MPO, a basic hallmark of neutrophilic inflammation causes protein carbonylation leading to oxidative injury in allergic inflammatory , and GST (F) activity: Activity of SOD, Catalase, and GPx in lung tissue was found lower and GR (D) activity was higher in TDI group than control and β-END significantly reversed their activity. Total reduced GSH and GST level were considerably modulated in TDI induced group and was significantly reversed as control with β-END treatment.
Results are represented as means ± SEM (*p < 0.05 Control vs. TDI group and #p < 0.05 TDI vs. β-END group). www.nature.com/scientificreports/ diseases 32 . Our present findings indicates that TDI exposure in mice lead to increased MPO activity, protein level and protein carbonylation whereas β-END inhibited MPO activity as well as diminished the protein and protein carbonyl content. Increased MPO might be correlated with cellular influx (neutrophilic and macrophage) in asthmatic mice leading protein carbonylation and further preventing inflammation by β-END administration.
Oxidative stress may play a crucial role as an initiating factor rather than acting as a concomitant aggravating factor in the pathogenesis of bronchial asthma and increase in ROS might overwhelm endogenous antioxidant defenses 38 . Considerable experimental evidence supports the idea that oxidant⁄ antioxidant balance in the airways is important for maintaining homeostasis in asthma and improvement in the antioxidant status may improve the inflammatory process 39 . In the ongoing study, the oxidant⁄ antioxidant balance was observed as a mean of TOS and TAS where oxidant/antioxidant imbalance was evidenced by an increase in TOS, and decrease in TAS level by TDI induction. The result was consistent with the study of Katsoulis et al. 2003 who also reported the lower level of TAS in asthmatic subject as compared to healthy subjects 40 . On the other hand, β-END reversely modulated thereby enhancing the TAS and suppressing the TOS.   www.nature.com/scientificreports/ Results are represented as means ± SEM (*p < 0.05 Control vs. TDI group and #p < 0.05 TDI vs. β-END group). The black arrow represents inflammation in alveolar areas; the red arrow represents inflammation in the peribronchiolar region; blue arrow represents inflammation in pulmonary blood vessels. www.nature.com/scientificreports/ Oxidative stress in asthma can be treated by two strategies including reducing exposure to oxidants and augmenting antioxidant defense 41 . In biological system, endogenous antioxidants defense includes SOD, Catalase, GR, GPx, GSH and GST to reduce stress generated by the free radicals and oxides. SOD and catalase, two crucial antioxidant enzyme functions simultaneously as an oxide radical scavenger 39 . SOD functions to eliminate the superoxide anion radicals by converting it into H 2 O 2 and O 2 , while catalse and GPx decomposes H 2 O 2 further to form water and oxygen 41 .GR, a glutathione regenerating enzyme play a critical role in GSH production by converting oxidised glutathione (GSSG) to reduced glutathione thereby acting as a reducing substrate in the redox cycle.Further, GST functions to inactivate various electrophilic substrate in conjugation with GSH and GR using NADPH as the reducing co-factor and thereby maintains an appropriate intracellular GSH level in the cell 42 . In the present investigation, the TDI asthmatic model expressed significant decreased activity of SOD, CAT, GPx and GST in lung tissues which may be associated with increased oxidants and oxidative stress and was concur with the earlier investigations 43,44 .Suppression of TDI induced airway oxidative damage was observed following the administration of β-END modulating the antioxidant enzymes as evident from increased SOD, catalase, GPx and GST indicating the antioxidant potential. GR helps in generating GSH and hence the level of both GR and GSH was excerbated in asthmatic mice and suppressed by β-END treatment. On the other hand, GSH has also  www.nature.com/scientificreports/ been reported to exacerbate the level of NO which has been linked with increased neutrophilic inflammation. GSH also protects the cell membrane from lipid peroxidation via the GSH-Px effect 34,44 . Lipid peroxidation, a mechanism provoked by free radicals leads to oxidative deterioration of peroxide radicals as lipid hydroperoxides and aldehydes as MDA 45 . In the present investigation, an increase GSH level might be correlated with increased NO and MDA content in TDI-induced mice whereas β-END declined GSH level as well as NO and MDA. Moreover, GSH forms also conjugate with different very reactive electrophilic compounds, through the action of GST and therefore GST level was also enhanced in TDI-induced mice but declined with β-END treatment.
Nrf-2 is a promising regulator of cellular resistance to oxidants and drives the expression of numerous cytoprotective genes involved in xenobiotic metabolism, antioxidant responses and anti-inflammatory responses. Nrf-2 regulates the basal and induced expression of an array of antioxidant response element (ARE)-dependent genes thereby regulating the oxidative stress-related genes and directly affecting the homeostasis of ROS and RNS 46 .
In an unstressed condition Nrf-2 is targeted for constant degradation by UPS resulting in a low level of free Nrf-2 protein and constraining the transcription of Nrf-2 dependent gene. However, cellular degradation of Nrf-2 in response to stress occurs through Keap1-Cul3-Rbx1 E3 ubiquitin ligase or β-TrCP-Skp1-Cul1-Rbx1 E3 ubiquitin ligase determining the keap-1 dependent or independent mechanism 18 . In the ongoing study, TDIinduced groups showed a significant increase in cytosolic Nrf-2, a decrease in nuclear Nrf-2, and an increased keap-1 as compared with the control group supporting previous study where TDI inhibited Nrf-2 translocation in the nucleus 47 . Further, β-END alternatively restored the level of both cytosolic and nuclear Nrf-2 comparable to control. However, keap-1 expression in the β-End group was similar to TDI supporting keap-1 independent regulation of Nrf-2 activation. It might be that the Neh6 domain of Nrf-2 has been phosphorylated by other proteins at serine/threonine residue resulting in disruption of Nrf-2 keap-1 interaction and supporting the keap-1 independent Nrf-2 activation. Several protein kinase pathways as phosphatidylinositol 3-kinase (PI3K), MAPKs, PKC, and glycogen synthase kinase-3 (GSK-3), c-Jun, N-terminal kinase (JNK) have been identified to be associated with Keap1-independent activation of Nrf-2 which needs to be further explored in the present study 48 .
Clinical and experimental studies suggest that a network of proinflammatory cytokines play important role in lung injury. TDI induces different immune response as a result of T-lymphocyte polarization towards T-helper Type 1 (T H 1) or 2 (T H 2) cells 49 . T H 1 lymphocytes secrete mainly IFN-γ and TNF-α promoting cell-mediated immunity whereas T H 2 cells support humoral immune response and are recognized by their secretion of interleukins as IL-4, IL-5, and IL-13 50 . The present study supports the significant increase in the level of proinflammatory cytokines TNF-α and IFN-γ in TDI-induced asthmatic animals, whereas β-END pretreatment had significantly attenuated TNF-α and IFN-γ level representing Th1 paradigm. TNF-α, representing pleiotropic activity is also reported for leukocyte recruitment including neutrophils determining asthma severity 51 . In the present study, the neutrophilic inflammation in the lungs can be concurrent with the TNF-α level.
Inflammation and oxidative stress results in lung injury further increasing permeability across the lungs, causing recruitment of immune cells, increase in size of alveolar space and damage of tissue walls as evident in histological slides. Histopathological studies of lungs reveal structural damage of alveolar spaces and tissue wall by TDI induction which was succesfully reverted by β-END treatment.
In summary the present study reveals for the first time the therapeutic role of β-END as anti-inflammatory and anti-oxidant in murine model of TDI-induced OA by exerting its effect via ROS and Nrf-2 inhibition.

Conclusion
In conclusion, the present study signifies opioid peptide, β-END to be a promising treatment for chronic asthmatic conditions by regulating inflammation, cellular apoptosis, and oxidative stress via Nrf-2 signalling mediators (Fig. 11). The work supporting the efficacy of β-END against chronic asthma are very preliminary and hence more research and exploration is required before β-END could be widely recommended as therapeutic. Experimental grouping. Mice were randomly divided into four groups (n = 7 mice/group) as in Table 2.

Methods
Group I were normal/control mice; Group II wereTDI induced mice which were sensitized and challenged with www.nature.com/scientificreports/ TDI as given in the protocol below; Group III were vehicle mice (TDI solvent); Group IV were β-END administered mice.

Development of TDI induced asthma.
TDI dissolved in olive oil and acetone (4:1) was used as an inducer for generating asthma in experimental mice. Mice were sensitized on day 0, 7, 14 with 1%TDI through intranasal route in both the nostrils in a volume of 20 μl (10 μl in each nostril). Further, from day 21 to 51 mice were exposed and challenged with 2.5% TDI thrice a week (alternate days) through intranasal route in a total volume of 20 μl (10 μl in each nostril) (Fig. 12).
Administration of β-END. β-END dissolved in saline was administered through intranasal route (10 µl in each nostril) at a dose of 5 μg/kg bw, 1 h before sensitization and challenge (Fig. 1). Dose of β-END was selected based on the previous studies 52, 53 .

Collection of samples as BALF and lungs.
Mice were sacrificed 24 h after the last TDI exposure and tracheostomy was performed. Trachea was cannulated and BALF was collected by washing the lungs with icecold PBS. Briefly, the lungs lumen was aspirated with 1 ml of ice-chilled PBS three consecutive times and a total volume of 2.5 ml of BALF was collected. Collected BALF was centrifuged at 2000 rpm and 4 °C for 10 min. The supernatant was collected and stored at − 80 °C for further assay of protein content, nitric oxide, and cytokine level (TNF-α and IFN-γ). BALF pellet was processed for ROS, total and differential cell count, and cellular apoptosis. Blood was collected by retroorbital bleeding in a heparin-containing tube and centrifuged at 3500 rpm and 4 °C for 10 min for serum. Serum was used to analyze nitric oxide levels. Lung lobes were aseptically removed for histological analysis for lung injury by H&E staining and other lobes were processed for TOS (Total oxidant status), TAS (Total antioxidant status), antioxidants enzymes, MPO, MDA and Nrf-2 and keap-1expression.  www.nature.com/scientificreports/ Total and differential cell count. The total number of inflammatory cells was determined by enumerating cells in hemocytometer by trypan blue dye exclusion test. Briefly, 10 µl of pellet was stained with 10 µl of trypan blue and placed on hemocytometer for counting. Further, 100 µl of aliquot was placed onto slides and cytocentrifuged (200Xg, 4 °C, 10 min) in a cytospun machine. Slides were air dried; fixed and stained using Giemsa stain. Cells were identified and counted on the basis of their nuclear morphology.  SOD activity. SOD activity was measured by previous described method of Das et al. 61 . Reaction mixture was prepared by mixing 1.14 ml 50 mM phosphate buffer (pH 7.4), 75 μl of 20 mM α-methionine, 40 μl of Triton X-100, 75 μl of 100 mM hydroxylamine hydrochloride and 100 μl of 50 μM EDTA. 50 μl lung homogenate supernatant was mixed with the reaction mixture and incubated for 5 min at 37 °C. Further, 80 μl of 50 μM riboflavin was added to the reaction mixture and incubated for 10 min inside a wooden box having light and coated with aluminum foil. After 10 min of incubation 1 ml freshly prepared Griess reagent (1:1 solution of 0.1% of NED and 1% of sulphanilic acid in 5% orthophosphoric acid) was added to the reaction mixture. Absorbance of the mixture was read at 543 nm and SOD activity was expressed as unit per milligram of protein.

Estimation of total oxidant status (TOS). TOS was determined in
Catalase activity. Catalase activity was measured according to the previously described method of with slight modification 62 . Briefly, reaction mixture was prepared by adding 490 µl distilled water, 1100 µl phosphate buffer (50 mM) and 500 µl H 2 O 2 (60 mM). Further, immediately 10 µl of homogenate supernatant was added and absorbance was measured at 290 nm. The decrease in absorbance was observed for 5 min. Catalase activity was expressed in µmoles/min/mg of protein. www.nature.com/scientificreports/ Assay of glutathione-S-Transferase (GST). GST level was assayed by the standard method as described earlier 65 . Briefly, 100 μl lung homogenate supernatant was mixed with 3 ml of the reaction mixture having 1 ml of 50 mM Phosphate buffer, 1.7 ml distilled water and100μln of 30 mM CDNB. The reaction mixture was incubated for 5 min at 37 °C. After incubation, 100 μl of 30 mM reduced glutathione was added and absorbance was measured spectrophotometrically at 340 nm. Enzyme activity was calculated in terms of μM of CDNB conjugate formed/min/mg protein.

Estimation of reduced glutathione (GSH).
Estimation of GSH was performed as per the established protocol 66 . Briefly, 100 μl lung homogenate, 600 μl reaction buffer containing 0.1 M sodium phosphate buffer (pH 7.0) and 1 mM EDTA were mixed. Further, 760 μl distilled water and 40 μl of 0.04%DTNB dissolved in 1% sodium tri-citrate were added. The reaction mixture was incubated for 5 min and absorbance was read at 412 nm. Using the standard curve, the GSH concentration for each unknown sample was determined and expressed as μM /ml.

Estimation of cytokines (TNF-α and IFN-γ).
The level of TNF-α and IFN-γ in BALF were estimated by commercially available ELISA kit as per the manufacturer's instruction (Biolegend, USA). The results were expressed in pg/ml.

Flow cytometry to analyze cellular apoptosis. Apoptosis was analyzed by labeling BALF cells with
AnnexinV (AV) and propidium iodide (PI) following the manufacturer's staining protocol to quantify the percentage of cells undergoing apoptosis, early and late apoptosis, and/or necrosis. BALF pellet cells were treated with lysis buffer (ammonium chloride and EDTA) to burst RBCs. Cells (1X10 5 ) were washed twice with cold cell FACS buffer (BioLegend) and then resuspended in AV binding buffer. In 100 μl of cell suspension, 5 μl of AV (FITC tagged) and 10 μl of PI were added and incubated for 15 min at room temperature in the dark. After 15 min 400 μl of Annexin V binding buffer was added and samples were applied to the flow cytometer to examine cellular apoptosis.
Lung morphology and severity. For lung histology, the left lung lobes were perfused with 10% Neutral Buffer Formalin (NBF), aseptically removed, and fixed in 10% NBF for 24 h. Lung tissues were sectioned (5 µm) from paraffin-embedded tissue and stained with haematoxylin and eosin (H&E). Pathological conditions were measured in the lung sections as a means of the degree of inflammation and injury (alveolar destruction) in all the groups under a light microscope. The degree of inflammation was scored by enumerating the layers of inflammatory cells in alveolar spaces, bronchioles, surrounding the vessel by two independent pathologists as per the earlier described method 68 . An overall severity score was calculated for each animal by adding the individual scores of inflammations, destruction, and emphysema and further represented by pooling and taking an average of the scores.
Statistical analysis. Experimental data are expressed as mean ± SEM (n = 7/group). Results for comparisons between multiple groups were observed for statistical analyses by applying student t-test and one way ANOVA followed by Turkey's test. Analysis was performed by SPSS software version17 (IBM, USA). The statistical significance was set at p values < 0.05 and < 0.001.

Data availability
The data presented in this study are available upon request to the corresponding author. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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